Strategies to enhance post-transplant T and NK cell reconstitution could decrease the incidence of fatal infectious complications and malignant relapse, and significantly improve the overall survival of recipients of a hematopoietic stem cell transplantation (HSCT). We propose studies in murine allogeneic HSCT models to analyze the effects on post-transplant T cell reconstitution of the adoptive transfer of ex vivo expanded T cell precursors to HSCT recipients. Large numbers of T cell precursors can be generated using a novel culture system using stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1). In preliminary studies we found that the adoptive transfer of OP9-DL1 derived T cell precursors to recipients of a T cell-depleted allograft can enhance early post-transplant T and NK cell reconstitution and donor chimerism. This improvement in post-transplant T and NK cell reconstitution results in significant graft-versus-tumor (GVT) activity without graft-versus-host disease (GVHD), and better antimicrobial activity against Listeria monocytogenes. Adoptive transfer of T cell precursors to HSCT recipients treated with keratino growth factor (KGF) had an additive effect on T cell reconstitution. We will assess the kinetics of T and NK cell reconstitution in recipients with varying degrees of MHC disparity, recipients with or without GVHD, young vs. middle-aged recipients, as well as the effects of varying doses of T cell precursors, the role of homing molecules, the effects of the timing of administration and multiple doses of T cell precursors, and the site of T cell development (thymic vs. extrathymic). In addition, we will test combination strategies using administration of T cell precursors and immunostimulatory cytokines/growth factors such as lnterleukin-7 (IL-7) and KGF. In addition to in vitro functional T and NK cell assays, we will assess the effects of T cell precursor administration to HSCT recipients on anti-microbial activity against Listeria monocytogenes and GVT activity in vivo. These preclinical studies could result in the development of a safe and effective adoptive cell therapy using transfer of T cell precursors in combination with cytokines/growth factors to decrease post-transplant morbidity and mortality from infections and relapse in HSCT recipients.